Recombinant Human CCL25

Recombinant Human CCL25 is used in research applications to study immune cell trafficking, inflammation, and cancer metastasis due to its specific role as a chemokine that binds to the CCR9 receptor, regulating the migration and activation of various immune cells.

Key scientific reasons to use recombinant human CCL25 in your research include:

• Immune Cell Chemotaxis and Homing: CCL25 is a potent chemoattractant for CCR9-expressing cells, including thymocytes, T cells, macrophages, and dendritic cells. It is essential for studying mechanisms of lymphocyte homing to the thymus and small intestine, as well as the recruitment of immune cells during inflammation.

• Modeling Inflammatory and Autoimmune Diseases: The CCL25/CCR9 axis is implicated in chronic inflammatory conditions such as rheumatoid arthritis, atherosclerosis, and endometriosis, where it mediates the migration of monocytes and macrophages to inflamed tissues. Recombinant CCL25 enables in vitro and in vivo modeling of these processes.

• Cancer Research: CCL25 promotes the migration and metastasis of certain cancer cells (e.g., melanoma, breast carcinoma) to the small intestine and other tissues expressing CCR9. It is also used to investigate tumor immunology, as CCL25 can enhance the infiltration of cytotoxic T cells into tumors, improving the efficacy of immunotherapies.

• Functional Assays: Recombinant CCL25 is used in bioassays to test chemotactic responses, cytokine secretion, and immune cell activation. It is suitable for migration assays, receptor binding studies, and immune cell polarization experiments.

• Controlled Experimental Conditions: Using recombinant protein ensures batch-to-batch consistency and eliminates variability associated with endogenous sources, enabling reproducible and interpretable results in mechanistic studies.

• Species Cross-reactivity: Human and mouse CCL25 proteins can act on human CCR9, allowing for translational studies between animal models and human systems.

Typical applications include:

• Chemotaxis and migration assays for immune cells.
• Investigating T cell development and thymic selection.
• Modeling tissue-specific immune responses and inflammation.
• Studying mechanisms of cancer metastasis and testing immunotherapeutic strategies.

In summary, recombinant human CCL25 is a critical reagent for dissecting the molecular and cellular mechanisms of immune cell trafficking, inflammation, and tumor biology, providing a controlled tool for both basic and translational research.

Yes, recombinant human CCL25 can be used as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and its concentration is accurately determined.

Supporting details:

• ELISA kits designed to quantify human CCL25 typically use recombinant CCL25 as the standard for generating the calibration curve. This allows for precise quantification of CCL25 in biological samples by comparing sample signals to those from known concentrations of the recombinant protein.
• According to general ELISA guidelines, a purified recombinant protein is suitable for preparing standard curves, as long as its concentration is reliably measured (e.g., by spectrophotometry or HPLC).
• The recombinant standard should be reconstituted and diluted according to the protocol, and the standard curve should cover the expected range of sample concentrations.
• It is important to confirm that the recombinant CCL25 is biologically and immunologically equivalent to the native protein, as most commercial ELISA kits validate their standards for recognition by the assay antibodies.

Best practices:

• Use the recombinant CCL25 standard in the same buffer and matrix as your samples to minimize matrix effects.
• Prepare serial dilutions to generate a standard curve, typically ranging from low picogram to nanogram per milliliter concentrations, depending on assay sensitivity.
• Validate the standard curve for linearity and reproducibility in your specific assay setup.

Limitations:

• Ensure the recombinant protein is not aggregated or degraded, as this can affect quantification accuracy.
• If using a recombinant standard from a different source than the kit manufacturer, confirm compatibility with your assay antibodies.

In summary, recombinant human CCL25 is appropriate for ELISA calibration, provided it is properly prepared and validated for your assay system.

Recombinant Human CCL25 has been validated for several key applications in published research, primarily in bioassays assessing chemotactic activity, cell migration and invasion, and functional studies of immune cell trafficking.

Validated Applications in Published Research:

• Chemotaxis/Bioassay:
  CCL25 is widely used to assess the chemotactic response of various cell types, including BaF3 pro-B cells transfected with human CCR9, thymocytes, macrophages, dendritic cells, and THP-1 cells. Its chemoattractant activity is typically measured in vitro using transwell migration assays or similar formats.

• Cell Migration and Invasion Assays:
  Recombinant CCL25 has been used to stimulate migration and invasion of cancer cell lines, such as non-small cell lung cancer (NSCLC) cells (A549, SK-MES-1), with effects quantified by counting migrated/invaded cells after treatment. These assays often include blocking experiments with anti-CCR9 antibodies to confirm specificity.

• Functional Studies of Immune Cell Trafficking:
  CCL25 is validated for studies on lymphocyte homing, particularly to the thymus and small intestine, involving integrin α4β7+ and αEβ7+ T cells, IgA-producing plasma cells, and γδ T cells. These studies often use recombinant CCL25 to induce migration or adhesion in vitro or in vivo.

• Inflammation and Disease Models:
  Recombinant CCL25 has been used in models of chronic inflammation (e.g., rheumatoid arthritis, atherosclerosis, endometriosis) to study its role in recruiting CCR9+ monocytes and macrophages, and in cancer metastasis models to investigate its effect on tumor cell migration and invasion.

• Adhesion Assays:
  Immobilized CCL25 is used to activate integrins and promote lymphocyte adhesion to endothelial ligands such as VCAM-1 and MAdCAM-1, relevant for gut-homing studies.

Typical Sample Types:

• Whole cells (primary cells, cell lines)
• In vivo models (mouse, humanized systems)

Experimental Readouts:

• Cell migration/invasion quantification
• Chemotactic index
• Adhesion assays
• Western blot for downstream signaling (e.g., VEGF, MMPs)
• Flow cytometry for cell surface markers

Summary Table of Validated Applications

Key Notes:

• Recombinant CCL25 is not typically used for Western blot as a target, but may be used as a positive control for functional assays.
• All applications are for laboratory research use only, not for clinical or diagnostic purposes.

If you require protocol details or specific assay recommendations, please specify the intended cell type or disease model.

To reconstitute and prepare Recombinant Human CCL25 protein for cell culture experiments, dissolve the lyophilized protein in an appropriate sterile solution, typically at a concentration between 100–250 μg/mL, using either sterile distilled water or 4 mM HCl, depending on the formulation and manufacturer instructions. Avoid vigorous mixing and repeated freeze-thaw cycles.

Step-by-step protocol:

• Centrifuge the vial briefly (20–30 seconds) before opening to ensure all powder is at the bottom.
• Reconstitution:
  • If the product contains acetonitrile/TFA, reconstitute in 4 mM HCl at 250 μg/mL.
  • For carrier-free or standard lyophilized protein, reconstitute in sterile distilled water or PBS (phosphate-buffered saline) at 100–250 μg/mL.
  • If recommended, add 0.1% BSA (bovine serum albumin) to the buffer to stabilize the protein and reduce adsorption to surfaces.
• Mix gently: Do not vortex or pipette vigorously; swirl or invert gently to dissolve.
• Aliquot: Divide the stock solution into small aliquots to avoid repeated freeze-thaw cycles.
• Storage:
  • Store aliquots at –20°C to –70°C for long-term use.
  • After reconstitution, the solution can be kept at 2–8°C for up to one month under sterile conditions.
• Working solution: Dilute the stock solution into cell culture medium or assay buffer to the desired final concentration (typically in the range of 1–180 ng/mL for bioassays, depending on cell type and experimental design).

Additional notes:

• Always consult the specific product datasheet for recommended reconstitution buffer and concentration, as formulations may differ.
• For cell culture, ensure the final buffer is compatible with your cells and does not contain toxic additives (e.g., acetonitrile or TFA should be diluted out before use).
• If using for chemotaxis or signaling assays, typical working concentrations range from 1–180 ng/mL.

Summary Table:

Always verify the specific formulation and recommended protocol for your batch of recombinant CCL25, as buffer requirements and concentrations may vary.